We have shown that the L-A dsRNA virus of Saccharomyces cerevisiae has, in its (+) strand, two open reading frames: ORF1, encoding the major coat protein (gag) and ORF2, encoding the viral RNA-dependent RNA polymerase and a single-stranded RNA binding activity (pol). We found that pol is expressed only as a gag-pol fusion protein, produced by a -1 ribosomal frameshift mechanism indistinguishable from that used by retroviruses to make their gag-pol fusion proteins. We developed frameshift vectors which are being used to screen for drugs affecting ribosomal frameshifting, a new approach to searching for anti-retroviral drugs. Either increasing or decreasing the efficiency of frameshifting adversely affects replication by the L-A proteins, presumably because of the altered ratio of gag-pol to gag proteins, and we isolated chromosomal mutants that dramatically increase th efficiency of frameshifting. We defined the domains essential for viral replication around the RNA polymerase consensus sequence in pol, anddellmit the location of the single-stranded RNA binding domain. In the replication reaction (in vitro synthesis of (-) strands on a (+) strand template) the polymerase first binds at an internal site on the template molecule (the internal replication enhancer) and then attaches to the 3' end where it the begins synthesis (a looping mechanism). We cloned and sequenced the SK12 and SKI8 genes, members of the yeast intracellular antiviral system that limits replication of the apparently unrelated dsRNA viruses, L-A and L-BC and the 20S RNA circular single-stranded RNA replicon. SK12 has homology with RNA helicases and ribonucleoproteins, while SK18 has a repeat sequence first described in bet transducin, but also found in other proteins. Our studies of the MAK10 gen show that it is repressed by glucose, and, unexpectedly, its control sequences are inside the coding region of an adjacent gene. MAK3 encodes a N-acetyltransferase necessary for acetylation of the N-terminus of the L-A major coat protein. The 20S RNA circular single-stranded replicon is apparently naked in the cell, since we found that what had been reported by others to be its coat w actually a heat-shock protein adventitiously co-purifying with the RNA. We showed that 20S RNA encodes a 90 kDa protein that is expressed in infected yeast cells and has some homology to RNA polymerases of (+) strand RNA viruses.g